Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi

doi:10.13560/j.cnki.biotech.bull.1985.2021-1616

Abstract

Abstract:

The roots，stems，leaves，flowers，and pericarps of Sapindus mukorossi Gaertn. are rich in bioactive triterpenoid saponin. In order to understand the expressions of related genes in triterpenoid saponin biosynthesis pathway，it is necessary to screen the stably-expressed reference genes. In this study，8 traditional reference genes including 18S rRNA（guanine1575-N7）-methyltransferase（Sm18S），actin-related protein 8（SmACT），elongation factor 1-alpha（SmEF-1α），large subunit ribosomal protein L1（SmRPL1），small subunit ribosomal protein S26e（SmRPS26），tubulin-specific chaperone C（SmTBCC），ubiquitin-conjugating enzyme E2 M（SmUBC12），E3 ubiquitin-protein ligase BAH（SmUBP）were selected as candidate reference genes based on the RNA-seq data of roots，stems，leaves，buds，male flowers，female flowers，and developing pericarps of S. mukorossi. The expressions of these candidate reference genes were detected by RT-qPCR in the root，stem，leaf，bud，male flower，female flower，and pericarps at different developmental stages，and the stability of them were then evaluated by geNorm，NormFinder and BestKeeper software and RefFinder online analysis tool. The results showed that the expressions of 8 candidate reference genes differed in the variations among all samples. And the optimal reference genes screened by geNorm，NormFinder and BestKeeper were also slightly different. The results of comprehensive analysis showed that SmACT，SmRPL1 and SmUBP expressed quite stably，while the stability of SmEF-1α was the lowest among all candidate reference genes. The RT-qPCR results of 8 triterpenoid saponin biosynthesis related genes using SmACT，SmACT+SmRPL1 and SmACT+SmRPL1+SmUBP as the reference genes respectively were in accordance with the results of RNA-seq. Thus，it is concluded that SmACT and the reference gene combinations of SmACT+SmRPL1 or SmACT+SmRPL1+SmUBP can be used as reference genes for gene expression analysis of triterpenoid saponin biosynthesis pathway in S. mukorossi and other biological processes in S. mukorossi and related plants.

Key words: Sapindus mukorossi Gaertn., reference gene, quantitative real-time PCR, triterpenoid saponin biosynthesis, expression analysis

XU Yuan-yuan, ZHAO Guo-chun, HAO Ying-ying, WENG Xue-huang, CHEN Zhong, JIA Li-ming. Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi[J]. Biotechnology Bulletin, 2022, 38(10): 80-89.

Figures/Tables 8

References 39

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内参基因 Reference genes	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequences（5'- 3'）
Sm18S	Samuk09G0136600	221	AGATTGAAGGCGTTCTTTGGAT	TCAGTGTTTATGGACGGTGGAC
SmACT	Samuk13G0061200	129	AGAAAGTTGGCCTCGCTGAA	CAGGAACCAGACCACCTGTC
SmEF-1α	Samuk14G0055700	101	TCCAAGGCCAGGTACGATGA	AAACCAGAGATGGGGACGAAG
SmRPL1	Samuk07G0011500	206	CTGACCACCCCACCACTCTC	TCCTCCTCATCTGCCACCTC
SmRPS26	Samuk08G0043900	230	GCGAAGAAATGGAGGAAGGA	AGTGGATGGCACACGAAACA
SmTBCC	Samuk01G0035500	130	AGCCTGACACCCTTTCTCTACC	CCAACCTCTTCTGCAGCCTT
SmUBC12	Samuk11G0037500	102	TTGTCTGGACCAACCAAAGGA	TGCTTCTTGCCAGGTGTTTTC
SmUBP	Samuk09G0089700	180	TGCACAGTTGTTGCTCATGC	CTGCTTCATTTTCCCCGTGC

内参基因 Reference genes	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequences（5'- 3'）
Sm18S	Samuk09G0136600	221	AGATTGAAGGCGTTCTTTGGAT	TCAGTGTTTATGGACGGTGGAC
SmACT	Samuk13G0061200	129	AGAAAGTTGGCCTCGCTGAA	CAGGAACCAGACCACCTGTC
SmEF-1α	Samuk14G0055700	101	TCCAAGGCCAGGTACGATGA	AAACCAGAGATGGGGACGAAG
SmRPL1	Samuk07G0011500	206	CTGACCACCCCACCACTCTC	TCCTCCTCATCTGCCACCTC
SmRPS26	Samuk08G0043900	230	GCGAAGAAATGGAGGAAGGA	AGTGGATGGCACACGAAACA
SmTBCC	Samuk01G0035500	130	AGCCTGACACCCTTTCTCTACC	CCAACCTCTTCTGCAGCCTT
SmUBC12	Samuk11G0037500	102	TTGTCTGGACCAACCAAAGGA	TGCTTCTTGCCAGGTGTTTTC
SmUBP	Samuk09G0089700	180	TGCACAGTTGTTGCTCATGC	CTGCTTCATTTTCCCCGTGC

内参基因 Reference gene	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequence（5'- 3'）
SmAACT4	Samuk13G0079600	132	CCTGTTTTGAGGGCATTGATTG	ACCAGCATAGAACGCAGCCA
SmDXS4	Samuk14G0066700	194	AACAATGCTCAGAAGCCGAGA	GCGATCCATCCAGTAATCCAC
SmFPS	Samuk04G0156300	131	GAGGCAGAAGTAGGAACTCACCA	CCATTGTAGCAGTCAAGTGGTAAG
SmbAS1	Samuk02G0323400	113	GAGTGGGATACTGGTTTCGCTAT	TCCTTGACCTGAGATGCTTTGA
SmCYP716A-5	Samuk08G0024600	146	GCTGTTTTCTGTGGCCCTTC	CGAGTCTTGGCAATTTTCCC
SmUGT73C-14	Samuk12G0007500	120	ACCTACAAGTGCCCAAACAGAT	ATTCCCAGTTCACCACATTCC
SmbHLH8	Samuk10G0068800	107	CAACCTTTGACTCCGACTCCA	CCTTCCCTTACCCTAACCTCC
SmERF25	Samuk11G0050000	129	CAACAGCCACACCAACAGCA	TCTTCTCGGGTCACGGATCTC

内参基因 Reference gene	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequence（5'- 3'）
SmAACT4	Samuk13G0079600	132	CCTGTTTTGAGGGCATTGATTG	ACCAGCATAGAACGCAGCCA
SmDXS4	Samuk14G0066700	194	AACAATGCTCAGAAGCCGAGA	GCGATCCATCCAGTAATCCAC
SmFPS	Samuk04G0156300	131	GAGGCAGAAGTAGGAACTCACCA	CCATTGTAGCAGTCAAGTGGTAAG
SmbAS1	Samuk02G0323400	113	GAGTGGGATACTGGTTTCGCTAT	TCCTTGACCTGAGATGCTTTGA
SmCYP716A-5	Samuk08G0024600	146	GCTGTTTTCTGTGGCCCTTC	CGAGTCTTGGCAATTTTCCC
SmUGT73C-14	Samuk12G0007500	120	ACCTACAAGTGCCCAAACAGAT	ATTCCCAGTTCACCACATTCC
SmbHLH8	Samuk10G0068800	107	CAACCTTTGACTCCGACTCCA	CCTTCCCTTACCCTAACCTCC
SmERF25	Samuk11G0050000	129	CAACAGCCACACCAACAGCA	TCTTCTCGGGTCACGGATCTC

内参基因 Reference gene	扩增效率 Amplification efficiency /%	相关系数R² Correlation coefficient R²	斜率K Slope K
Sm18S	97.872	0.998	-3.374
SmACT	93.727	0.999	-3.482
SmEF-1α	99.789	0.999	-3.327
SmRPL1	100.206	0.999	-3.317
SmRPS26	94.469	0.999	-3.462
SmTBCC	97.196	0.997	-3.391
SmUBC12	96.881	0.999	-3.399
SmUBP	98.032	0.999	-3.370